Biochemical processes for the oxygenation of steroids



Patented Jan. 12, 1954 BIOCHEMICAL PROCESSES FOR rHE OXYGENATION Ovr-mews .Osear. Ii Worc Rob rt- P-- .Jac senl .Shmws urt,

and Gregory P ncus, w M Schenker, Shrewshury, Mass, and CharlesMarshall, Chicago, 111., assignors to G. D., Searle Ro J 12 Harold Levy,

1e QQ-z cheese 11 a rtfl sti 9f i hw i N Drawing .AnnicationJanuary"25,1351,

fie ial- 2,411,826 11 s am -119 .4

lCHeOH- H s GO nt orti sterve he r v la le. on y in amq mits,i man ma'ad ens gl ds-j 7 Cbrticostergne omen the eby i r fx e .l'I-Qvdmmcofl cotmm 9 the which isv one of themost important and pharmacologicallyuseful hormones of the adrenal gland, but-likewise heretofore availablein only very minute amountsfrom natural sources.

Certain othersteroidscanbe bio-oxygenated by the methods of invention,such as androsterone which forms 1lfi-hydroxyandrosterone; 4-

androstene-3-,17 -d-ione' similarly treated forms the correspondingllp-hydroxy derivative. Pro gesterone by-themethods of our invention isconvertedinto a mixture of l7-hydroxycorticosterone, corticosterone, Ii8-hydroxyprogesterone, 1 7- hydroxycorticosterone and other sterols.Other steroids which can be bio-oxygenated include 17-hydroxyprogesterone, 1-1"-desoxycorticosterone -acetate, -11 -desoxy 1 7--hydroxycorticosterone 21- acetate, isoandrosterone,.dehydroisoandrosterone, 1 6-dehydropregnenoione, M-androstehe-B, '11-dione, 'A pregnene 17,20,21 triol 3- one, A pregnene-3 ol-20-one andrelated pregnanediols and pregnanolones; Solubilized derivatives ofthese steroids, such as glucosides, hemisuccinates; and-the l-ike, canalso'be-used.

' By the-methodsof this invention it is also possible to ll-bio-oxygenate functional derivatives of the alcove-mentioned steroidssuchas the acetate .or propionate. Thus -1-1-desoxycorticosterone-21-acetate andits corresponding 17a.- hydroxy derivative are convertedbythe methods of this invention into corticosterone and 17- hydroxycorticostercne respectively, the acetoxy group-beinghydrolyzedduring-the perfusion and isolation processes.

It willheapparent to one skilled in the art that .th i I "'hY. Q s e pdsi qrni b the of this inventionmay 'bereadily convertedby lgnovvnmethods to the corresponding phar- Inacol'ogically useful l l-oxo(oxidation) 'or 11,I 2 or 9) anhy clro (dehydration) compoundswhereasth'e reverse transformations, reduction of '11- oxo or'hydrationof 11,-(12 or 9)"-anhydro to 11poxy compounds are extremely diflic ultor imposbible to carry out.

aceaoic Thus it can be seen that the processes or this invention forintroducing the llB-hydroxyl group into steroid hormones of the adrenalcortical type are of unique importance. llfi-hydroxy steroidal hormoneslike corticosterone and 17- hydroxycorticosterone, and related hormonessuch as 17 hydroxy ll dehydrocorticosterone and ll-dehydrocortioosteroneafiect the carbohydrate and protein metabolism by increasing the liversstore of glycogen. These hormones also increase the working capacity ofisolated muscle. A most important use forl'i-hydroxyll-dehydrocorticosterone has been in the treatment ofrheumatoid arthritis. The use of this substance in rheumatoid arthritishas resulted in improvement within a few days. Pain, stiffness andlimitation of movement were reduced. and tenderness and muscular painrelieved. Appetite and weight improved and toxicity was replaced by asense of well-being. The role of the hormone in other conditions such asmyasthenia gravis and rheumatic fever is being studied.

It is the object of this invention to provide simple and relativelyinexpensive methods for obtaining these llc-hydroxy steroids and theirderivatives from accessible starting materials. It is further an objectto provide methods for producing l'Y-hydroxycorticosterone and relatedllp-hydroxy compounds in a state of purity by simple and efficientbiochemical methods. Another object is to provide methods of producingllfi-hydroxy steroids, such as 17-hydroxycorticosterone, which can bereadily oxidized to ll-keto steroids, such as cortisone, byaeetylationof the Zl-hydroxyl radical followed by mild chromic oxideoxidation of the ll-hydroxyl radical. Other objects will be apparent tothose skilled in the art.

According to our invention, excised mammalian adrenal glands aremaintained in a functioning state by perfusion with an appropriatemedium, natural or synthetic, at temperatures in the range of -40 C. andpreferably at 3637.5 C., and

to the perfusion medium is added a quantity of steroid to behydroxylated. After the medium has circulated through the gland at leastonce, the perfusate is removed and the hormones separated by suitableprocedures. The perfusion medium can comprise blood, blood plasma with Usaline, or saline solutions of gelatin, technical blood albumin,synthetic plasma substitutes, and the like or mixtures thereof. For mostsuccessful operation, homologous plasma or whole blood or mixturesthereof constitute the most desirable perfusion media. Whole blood,before or after hemolysis or defibrination, or saline-diluted mixturesthereof are further preferably treated by being circulated through aliver preparation before use as a perfusion medium, though for shortperiods of perfusion this may not be necessary. This is desirable bothin order to remove and to prevent formation of clots, and to removevasoconstrictor substances present in these media which mightinterfere'with the free flow of the perfusate through the organ. Theperfusion medium may be supplied to a single gland under eitherpulsating or non-pulsating pressure or to a parallel bank of severalglands under nonpulsating hydrostatic pressure. The pressures used havebeen in the range between 20 mm. of mercury and 200 mm. Generallypressures in the range of 20-100 mm. are preferred. The pulsations usedhave been at a rateof about 24 to 180 per minute. Whereas pulsatingpressures 4 may be more physiological for the perfusion, steady hydrostaticpressures are also satisfactory. The length of the period of perfusionmay vary from a fraction of an hour to several hours depending upon therate of flow and the volume to be perfused. It is only necessary thatthe fluid containing the steroid pass through the functioning gland atleast once. Additional cycles are useful for producing more of theoxygenated steroids particularly at the higher input concentrations ofprecursor. In practice we have found that the more rapid perfusions (0.5to 7 liters/ gland/ hour) are quite satisfactory both as to conversionrate and purity of product; with faster flow rates the number of cyclesmay be increased. The concentration of the steroids in the perfusionmedium may vary from 50 to 1000 mg. per 1000 ml. of perfusion medium.The preferred'concentration is about 50-300 mg. per

liter.

The adrenal glands which are used in our processes may be obtained fromany of the conimon animals including the horse, sheep, pig, cattle, dog,rabbit, cat and monkey; those from the larger species are mostconvenient to use. The following illustrate the me'thods'of preparingmammalian adrenal glands for use in our procedure: A. Intact glandarterial flow.--A laparotomy is performed on an anesthetized or freshlykilled animal and the kidney region exposed. Theen tire region of thepaired adrenal glands is dissected. Two glands are removed from thekidneys of each animal, care being taken not to slash the gland. Thegland is dropped at once into cold saline solution and used as soon aspossible. Extraneous fat and tissue is removed from the gland and smallvenal entries near the main large vena cava are tied oif. A cannula isinserted into the aorta just below the renal arterial branch supplyingthe adrenals or into any direct aortic branch to the adrenal region, andall of the extra-adrenal arteries are ligated. A buffered balanced saltsolution may be washed through the cannulated preparation to remove allblood remaining in the vessels of the preparation but this is notabsolutely necessary. The cannula is then affixed to the arterial entryof a perfusion pump designed to circulate the perfusion mediumcontinually under either pulsating or nonpulsating hydrostatic pressureas specified above, and circulation of this medium is promptly started.It is essential that transfer of the cannulated preparation to theperfusion apparatus be as rapid as possible and that prompt perfusion bestarted with the medium at a pressure sufficient to insure activecirculation through the glandular tissue. The above operations andperfusion are best conducted under completely sterile conditions inorder to insure maximal survival of the glands, though for shorterperiods of perfusion absolute sterility is not required. Practicalsterility can be maintained by the addition to the medium of suitableconcentrations of antibiotics such as penicillin and streptomycin.

B. Intact gland venous flow-The dissected adrenal obtained as in A istrimmed to remove all of the extra-glandular tissue. A suture is loopedaround the neck of the adrenal vein and a cannula is pressed in andtied. By flooding the cannula with saline solution while massaging thegland, the system is freed of air. Saline supply under low pressure ispassed through to flush out the gland. During this time all fatunemmanousaissue and the major residueof the wall oii the vena cava are'trirnmed oil. The g land is tii'enstored 1 in l cold-saline solution.The cannulat'ed preparation', is then washed with saline andtheicannulaamxed to a perfusionapparatus as above.

C. Lacerated gland venous flow.The trimmed 'g-lan'dwith venouscannulation as in B-is-su-bjected ='--to multiple cortical laceration=ove'r vir tually its'entiresurface, the cutsbein'g spaced m5to' 3-mm.'-=apart and*made to a depth-of about Mite /g' the' thicknessofthe-cortical layer. Careis taken to lacerate the cortical layer only andtoavoid' the 'deeper modular-layer. When lacer ation' 'is'complete-saline solution under a-pres-- sure of zo scmmpis applied tothe cannula to chech adequacy 'off fiow. "Following laceration the glandis attached to :the perfusion apparatus, care being taken to avoid airbubbles. 'lheperfusion medium is passedthrough the gland and thasolutionof thesteroid is added to the: supply reservoir of.fluid. During theperfusion pressuresof 2'0'to 70 mm. are maintained throughoutandoxygenis continuously fedv into the perfusion liquid. Thistype ofpreparation is particularly usefulin banks of several glands eachattached to. a branchof a manifold type of perfusion apparatus.utilizing .nonpulsating flow under hydrostatic .pressure.

"j'lhesteroidi hormones which are produced by our, processescan beisolated from the perfusion medium by a variety of methods. The simplestis'b'ydir'ect adsorption with charcoal followed by elution of thecharcoal with suitable solvents such asmethylene chloride, ethylenechloride, benzene, chloroform, acetone, Q methanol, ethyl acetate. andthelike. .Upon removal of the sol vent, .preferab ly 'by evaporationunder vacuum atjlowtemperature. in an inert atmosphere, there isobtained.a residue of the steroids. This residue'is contaminated with a smallfraction of the lipoidalmaterial normally present in blood.Alternativelythe perfusion medium may be dial'yzedxagainst salinesolution and the latter treated. with charcoal as above (or during thedialysis) or extracted directly with a solvent such-as chloroform,methylene chloride, ethylene chloride'andthe' like. The steroids whichare in solution in the perfusate pass through the membrane and are aboutequally distributed between themedium andthesaline solution. Withcharco'aliinthe salineduringdialysis the-steroids are rapidlyadsorbed'and the dialysis period shortened; This method has the advantage-oravoidingccontaminationwith lipoidal material, but is time-consuming. Inthe most convenient isolation procedure, the hemolyzed perfusate isstirred each'day with about 5 g. per liter of activated charcoal for 3to 6 days, the blood being decantedafter. settling of the charcoal. Insmall scale'experimentsthe charcoal portions are combined andstored insaline at about 5 C. during the treating period, while on a larger scalethe individual charcoal portions may be collected at once, washed withwater or saline and dried. Thedriedcharcoal is then exhaustivelyextracted with warm solvent disclosed above, such-as methylene chloride,and the desired .ll-oxygenated steroidslare. isolated in a relativelypure, semicrystalline form. Further extraction of the charcoal with-warmbenzene, or other suitable solventsmentioned above, maybe resortedto ifnecessary,- to remove any remaining material. Theyproducti'may-befurther purified by direct crystallization or by chromatographicresolution:

The preferred adsorbant for 'the latter :jmet-hod is silica gel infinely divided fform.

As used herein, the term oxygenation refers to the introduction of anatom-oi oxygen-into an organic compound, resulting in the formationcof ahydroxylradical. A tormaldehydogenic steroid is one which containsa'n-a-ketolio'r a-glycolqgroup' at the 20-21-position, whichon-pei'ioliieacid oxidationgives r ise -to formaldehyde. Our inventionis illustrated in":detail b z-means of thefollo'wingexamples but is'notto Ice-construed as limited thereto. In' these examples; volumes arereported as liters (1.) and milliliters (ml.), weights as grams (g.) andmilligrams (-mg.) pressures as millimeters of mercury, v(mm), andmeltingpoints as degreescentigrade. 0.). The term .citrated-blood meansvolumesrof whole-blood mixed with 15 volumes of citrate solution (anaqueous solution containing 1.6% sodium citrate and 5% glucose). Theterm .citrated saline solution means an aqueous solution containing 0.8%sodium chloride and 0.35% sodium citrate. Saline solution means asolution of sodium chloride of 0.8 to 0.85% concentration.

Example 1 To a mixture of 500 cc. of plasma and 500 cc. of whole bloodwas addedZcc. of propylene. glycol containing mg. ofll-de'soxycorticosterone and the resulting solution was perfused witharterial fiOW through an intact beef adrenal-gland at an average flowrate of about. 0.5 liter per hour and a pulsing pressure of about-100mm. orm'ercury. l"l1e perfusate was .then hemolyzed pby freezing andthawing, stirred for.30 minuteswith 5 g. of activated carbon at roomteinperature'and allowed to settle for several hours. The blood wasdecanted and the charcoal-treatment repeated twice during-a period oftwo days. The combined charcoal was collected, washed .-with salinesolution and dried-by washing with a little acetone. The air-driedmaterial was then-exe tracted in a Soxhlet apparatus with methylenechloride for twenty hours and the extract concentrated under diminishedpressure undernit rogen. The residue consisted offal mg. ofsemicrystalline material containing 23 mg. ot-formahdehydogenic steroids(The Hormones, by Pincus and Thimann, vol. I, 1948, p.613). The combinedresidue from 5 liters of perfusateprocessed as above was chromatographedon silicagelby -elu-, ting with benzene-ethyl acetate mixtures providing64 mg. of crude crystalline corticosterone and 70 mg. of unaltereddesoxycorticosterone.

The corticosterone fraction, after purification byrecrystallization fromethyl acetate-neohexane or :ether-acetone-hexane, melted at. 1'73.3-180C. (powder) or 178-186.? C. (intactcrystals). The rotationin ethanol was[a] =+227 (c. '-0.*240).; Mixed withauthentic corticosteroneisolatedfromnatural sources, M. P. 173-179". C. (powder) or. 176.5-181C. (intact crystals), the perfusion product mixture-melted at 173-l81" CAcetylation of the perfusion product provided an acetate melting at1505-1525 (3., .whose mix-- ture I with authentic corticosterone2l-aoetate,- M. P..l51-.5-152.5Q., meltedat- 150.51152 ;C.

Example 2 A solutionof 100: mg .of' desoxyco'rtico'sterone in 400. cc.of whole blood was perfused with arterial 'flow through an-intact beefadrenal gland The saline solution was then extracted with chloroform andthe solvent layer washed, dried and concentrated under diminishedpressure under nitrogen. Analysis of the semi-crystalline residue by theperiodic acid method indicated the presence of 21 mg. offormaldehydogenic steroid from the 100 mg. of desoxycorticosterone used.This material was assayed by the glycogen deposition test (Pabst et al.,Endocrinology, 41: 55-65, July 1947) and was found active at the 500microgram dosage level. This material was therefore approximatelyequivalent to 10 mg. of corticosterone.

Ezrample 3 A solution of 150 mg. of desoxycorticosterone in 1500 cc. ofplasma was perfused as in Example 1 for about 3 hours. The perfusate wasthen placed in viscose tubing and allowed to stand at 2 C., with gentlerocking, for about 2.5 days with 500 cc. of citrated isotonic saline(0.80% sodium chloride, 0.45% hydrated sodium citrate) containing 1.5 g.of activated charcoal. The charcoal was collected, washed with. water,and extracted with acetone and benzene. The solvent solutionswereevaporated under reduced pressure in a nitrogen atmosphere and theresidue extracted with chloroform. The chloroform solution was driedwith anhydrous sodium sulfate and evaporated under vacuum. The residueof 83 mg. of semi-crystalline material contained 61 mg. offormaldehydogenic material. This was chromatographed on silica gel as inExample 1 providing 25 mg. of crystalline corticosterone of practicalpurity melting above 160 C. and 25 mg. of unaltered desoxycorticosteronemelting above 130 C.

Example 4 A solution of 250 mg. of desoxycorticosterone acetate in 2500cc. of plasma was perfused as in Example 1. The perfusate was treatedwith 25 g. of activated carbon in two lots at room temperature duringabout ten hours. The charcoal was washed, dried, and extracted withacetone, methylene chloride and benzene. The bulk of extracted residue(193 mg. out of 256 mg.) was obtained from the methylene chlorideextract and was found to contain 164 mg. of formaldehydogenie material.This was chromatographed on silica gel providing 24 mg. of crudecrystalline corticosterone and 51 mg. of unaltered desoxycorticosterone.

Example 5 A solution of 100 mg. of 17-hydroxy-11-desoxycorticosterone in1000 cc. of citrated isotonic saline containing hemolyzed blood wasperfused with venous flow through a laceratedbeef adrenal gland usingpulsating pressure for 1.5 hours (8 cycles). The perfusate from two suchperfusions was treated at room temperature with 40 g. of activatedcharcoal over a three-day period. The combined charcoal was collected,washed, dried, and exhaustively extracted with methylene chloride. Themethylene chloride extract was evaporated under nitrogen at reducedpressure. The extract residue was dissolved in warm ethyl acetate andchilled. Crystalline 1'7- hydroxycorticosterone was thus obtained. Themother liquors were evaporated under nitrogen at reduced pressure andthe residue was taken up in benzene and chromatographed over silica gel.The l'l-hydroxycorticosterone was eluted with benzene-ethyl acetate.There was obtained a total of 32.5 mg. of l'l-hydroxycorticosterone ofpractical purity melting above 200 C. together 8. .with a trace ofunaltered starting material. The, product after furtherrecrystallization fromethyk acetate melted at 207-209 C. The mixedmelting point with authentic 17hydroxycorticosterone: isolated fromnatural sources was not depressed.

Emmple 6 A solution of 600 mg. of desoxycorticosterone in 3000 cc. ofwhole blood was perfused with venous flow through a lacerated beefadrenal using non-pulsatile hydrostatic pressure for three hours (1cycle). The perfusate after hemolysis was treated over a three-dayperiod at roomv temperature with 60 g. of activated charcoal. Extractionof the charcoal and chromatographyof the extracted residue provided 241mg. of corticosterone of practical purity melting above 160". C.together with mg. of unaltered starting material.

Example 7 A solution of 2'70 mg. of androsterone in 2700 cc. of plasmawas perfused with arterial now through an intact beef adrenal gland for6 hours using pulsating pressure. The perfusate was. treated with 40 g.of activated charcoalover a three-day period. The charcoalswere'combined, washed with saline solution and then extracted withmethylene chloride for 16 hours in a Soxhlet; apparatus. Evaporation ofthe extract under nitrogen at reduced pressure provided 405 mg. ofsemi-crystalline material. This residue was dissolved in benzene andchromatographed on silica gel. The column was developed with abenzeneethyl acetate mixture (20:1) and then the androsterone fractionmg.) was removed by ben zene-ethyl acetate (4:1). The column was theneluted with benzene-ethyl acetate (2:1) and this eluate was evaporatedgiving a residue of 18' mg. of ll-hydroxyandrosterone in,non-crystalline form. This residue was dissolved in pyridine andtreated with acetic anhydride to form ll-j hydroxyandrosterone3-acetate, which is a crystalline ester. This material wasrecrystallizedfrom ether and melted at 238240 C. It. was found to beidentical with an authentic. sample of ll-hydroxyandrosterone 3-acetateby mixed melting point and by infrared absorption.

spectra.

Example 8 A solution of 750 mg. of desoxycorticosterone in 1000 cc. ofplasma containing 20% hemolyzed blood was perfused with venous flowthrough a lacerated beef adrenal gland using pulsating pressure for 45minutes (2.5 cycles). The perfusate was treated at room temperature with20 g. of activated charcoal over a three-day period. The materialextracted from the charcoal with math ylene chloride weighed 732 mg. andafforded. after chromatography on silica gel, 82 mg. of corticosteroneof practical purity melting above 0. together with 407 mg. of unalteredstarting material. I

Example 9 300 mg. of ll-desoxycorticosterone acetate in, 16 cc. ofpropylene glycol and 8 liters of citrated blood as in Example 17 wasperfused with arterial flow through an intact beef adrenal gland for 10hours (one cycle). The perfusate was frozen, thawed, and treated withactivated carbon. The carbon was washed with saline and acetone. thenextracted in a Soxhlet apparatus for 20 hours with methylene chloride.The methylene ch10 ride extract was evaporated under nitrogen in vacuo.The: residue: was. dissolved in benzene and passed rover: 18cc; f:silica-gel: .The ,cc lumn; was developed with benzene-ethylnacetatemixtures as in Example 17. There were thus obtained 24 mg. ofcorticosterone melting at 168-172 C. and 95 mg. of desoxycorticosteronemelting at 13.4:-1.38 C., after recrystallization from ethyl acetate.

.Excmple .10.

A solutionof 200mg. of desoxycorticosteroneiin 2000 cc.of'citratedisotonic saline eontaining6% of technical blood-albuminand-2% of hemolyzed fresh blood. was perfused. with. venous flow througha lacerated beef adrenal gland using pulsatingipressure for abouttwohours (6' cycles). The perfusate was treated with charcoal and the laterextracted afiording 377 mg. of semicrystalline residue. Thiswaschromatographed on silica gel yielding 71-. mg, of corticosterone ofpractical purity melting above 160- 0. together with; 39 mg. ofunaltered starting material.

' Example 11 Example 12 Bliters of-"citrated; filtered, whole" bovineblood were perfused'through two rat livers to remove fibrin. 'I?o,,the,.v blood wassadded' penicillin and streptomycirr'solution (800,000units of penicillin G and-"800'- mg of" streptomycin sulfate in 80 ml.of saline solution) 'and'then22 ml. of apro:- pylene glycol solution,containing 551 mg. of progesterone.

The mixture was then perfusedv Each: acetone wash: or extractwaszconcentrated in vacuo to remove almost all of the acetone, and theresidualzliquid. whichwasmainly water was extracted with fiveportions'of chloroform. The, combined chloroform extract wasconcentrated inyacuo to a weighed residue.

Each activated carbon preparation was then washed copiously on thefilter with methylene ch ri andfinal xtr ctedv with th ene chloride irra,Soxhlet. apparatus for. 16-24 hours. The, methylene. chloridesolutions, from each. per! tion of activated" carbon were combined andcon.- centrated in. vacuo to Weighed residues, After the methylenechloride extractions, the first batch of. charcoalwas combined with thesecond, the thirdlwiththe fourth and the fifth was. combined'with thecharcoallfrom the saline washes, andeach ofjthethree resulting charcoalprepara tions. was. extracted continuously with benzene. for 3-5 hoursusing, amodified Soxhlet apparatus. The modification consisted instirring the charcoal during the extraction and using a plug ofglasswool asafilter.

From the. six charcoal preparations described above the following.weights. of. material .were obtained by the variousextraction-procedures described above. Each of these residues was testedwith concentrated. sulfuric. acid: for green fiuos rescence, which is anin'dication of llehydroxy steroids such as corticosterone' and17-hydroxy corticosterone. The following table indicatesthe weights ofmaterials obtained from the various charcoal fractions by extraction andtheresults obtained on the concentrated-sulfuric acid test:

Acetone 3 I Batch of extract Fluorescence gg g gg Fluorescence. Benzene.FluorescencedarbO-n (viachlowvjn cone; extract th cone. extract, withcone.

rofglrgm), 112804 H2304 mg. H2SO4 112 Stron een 622 St on 12g .;.d 39249 shghtgreen" reen 93 -'ace- 2% Tree; Neggtive 59. 5 Negative.

o V o 16 -Negative I .42 'do through four laceratedbeef adrenal glandsin holllffiz allowingctha charcoal :to. settle overnight at, room;.tcmnerature and then separating the su ata t: bloods. Ira-decapitati nand 56611313101511? The. thimblesand. filter papers. used. in theextractions above'were combined and extracted by boiling with twoportions of benzenaeachfor 1 hour. The-combined benzene extracts'wereevaporatedin vacuo. givingaresidueof 23.5 mg. which gavenofluorescencewith sulfuric acid. The total weight of: extracted:materialfromall ofthe fore.- going procedurestwas 283.7 mg.

The third, fourth, fifth, and sixth methylene chloride extract residues.in the above table were combined with the third benzene extractdissolvedinbenzene and passed over a column containing 30 g. of silica gel passesthrougha IOU-mesh screen and'9 '0%..is retained on a 200- mesh screen).The column of silica gel was et Eachrcharcoal pgrtion wagthemwashed '25thendevelopedby-treatment with'benzene-a-nd benzene ethyl acetatemixtures according to the following table:

7 7 v 1 Weight H so cum 1 a Ehmte (ml) solvent residue test 1 80 Benzene132 2 100 Benzene-ethyl ace- 687 tote (30:1). 3 100 Benzene-ethyl ace-182 tate (:1).

4 100 Benzene-ethyl 2100- 58 Negative.

tate (10:1 5 100 Ethyl acetate 115. 5 D0- 6 100 do 14 Greenfluorescence. 7 100 do 8 Weak fluorescence. 8 100 Acetone 28 Slightfluorescence. 9 100 Methanol Each of these eluates was evaporated invacuo and each of the residues was given the sulfuric acid test. Theresults of these tests are indicated in the above table.

A second chromatogram was conducted as fol- .lows. The charge consistedof the'first and secand methylene chloride extract residues. weredissolved in benzene and passed overa column containing g. of silicagel, as above. column was then eluted with benzene, benzeneethyl acetatemixtures, ethyl acetate, acetone and These The methanol according to thefollowing table:

1 2 A fourth chromatogram was conducted using as the charge the benzeneextracts and 10 g. of silica gel. The following results were obtained:

v1 weirght H53 0 um 0 2 4* Eluate (ml.) solvent residue test 16 Benzenev42 v l5 Benzene-ethyl ace- 15. 5

tote 10:1). 15 Benzene-ethyl ace- 2.5

tate (9:1). 4X15 Benzene-ethyl ace- 61 1 Y tate 2X15 Benzene-ethyl ace-2 tote (7:1). 2X15 Benzene-ethyl ace- 1. 5 2X15 Benzene ethyl ace- 43X15 Betngene-ethyl ace- 5 Negative a e 2X15 Benzene-ethyl ace- 3 D0.2X15 Bengenethyl ace- 3 Do.

a e 2X15 Benzene-ethy1 ace- 5. 5 Trace.

tate (1:1). 2X15 Benzeneethyl ace- 1. 5 Negative.

tate (1:2). 2X15 Benzeneethyl ace- 2. 5 Strong fluotate (1:3). rescence.50 Ethyl acetate. 7 Slight fluorescence. 50 Acetone 6. 5 Negative. 50Methanol 7 Do.

The fifth chromatograph was conducted on 17 g. of silica gel using asthe charge the residues Weight fr 1 t 5 d 24 d b d Th 1' volume om e uaes an escri e above. e to Solvent ofresi- 1180 test mum (m1) due mg) 4lowing results were obtained. 1"

10 i 80 Benzene '52 11 100 Benzene-ethyl ace- 8 12 100 state 31 119 v 1weirght nso tate (20:1). 40 must" (1111.) 5mm residue 12...; 13 e. 100Benzene'ethyl acc- 60 (mg mm 10:1). 14 100 Ethyl acetate 416 Green fluo-Iescellce- 61 30 Benzene 2. 5 138 D0- 62 25 Benzene-ethyl ace- 3 '18.5Do. tate(10:1). 8 DO- 63 25 Benzene-ethyl ace- 1 41 Do. tate (91).Methanol 11 5 64-65 2X25 Benzelgeishyl ace- 20 66-70 5x25 e n zei 'e gimacees a e 2 Each of the various eluates was evaporated in 71.73 3X25Benzerze-eghylacevacuo and the residue given the sulfuric acid test. me6:

74-76 3 25 Ben ene-ethl ce- 15.5 Crystallization of the residue fromeluate No. 15, X a. a which weighed 138 mg., led to 22 mg. of crystal-7- 3X Benzenejethvl ace line I'Z-hydroxycorticosterone melting at 200-0. 5X25 z n le i 201 5 C tate (3:1).

85-87 3X25 Benzene-ethyl ace- 14 A third chromatograph was carried outusing me as the charge of the six acetone extracts. These 88-90 3X25iggg i 322 332}- combined residues were dissolved in benzene and 91-97 e2s,100 Be lngene-ezthyi ace- 41.5 See note. 2. G I passietd over 18 g.of silica gel Wllih the fOllOWmg 100 t ,fi 3 Negative,

u s: a e res 100 Ethyl acetate. 3 100 Acetone 16 100 Methano1 6 Weight Hso Eluate 781F315 Solvent 5a g 4 Norm-Residues 91 and 92 gave a slightfluorescence. 93-95 gave 1 a strong fluorescence. 93-97 were negatlve.

20 Benzene Negame- Crystallization of the residues 81 to 84 whichBenzene-ethyl ace- 34 Do. 21 mm (30.1), weighed 34.5 mg., from ethylacetate led to 3.2 22 100 ggg fl mg. of crystalline steroid of unknownstructure 23 100 Benzen ethyla melting at 202-205 C. The crystallizationof 24 100 E1??? ice't c'e 138 Do. 0 residues 93 to 95, weighing 18.5 mg.from a mix- 25 100 igga g ture of ethyl acetate and neohexane, led totwo 3.5 150. crops of crystalline materials, the first weighing 9.5 mg.and melting at res-197.5" c. and the sec- 100 Methanol 25 0nd weighing2.9 mg. and melting at 191.5-199 C.

13 *afiDJZdC'CIECEJZ-E mg: 01: 117+hydroxycortieosterone mltingat207fl-208i5." Ci. ..A:sixthzrchromatogramzwas:conductedmsing;as .thezchargezthezresidues from eluates fi, .7, 14; '15 (except for the 222mg::of: l7-hydroxycorticose terone obtained from residue 15), 16, 17, 25,26,

and 27. These combined residues were dissolved -irrhenzeneand"passedover-a column:of- 37:5g; of silioa gel;

acetate, acetone, and methanol with the- 'follow= 'ingresultsr Weight'Volume' of H550 (ml) Solvent test 4 residue Benzene:

Benzenee" tate:(10:

; Negative.

Do. Do. Do;

Dov Do. v Dot "Draco" .and f l u' o r e s cence.

Do.- Slight fiuorescence.

o. .Greenfl-uorescence.

Do. Slight .iluorescence.

Benzene-ethyl" ace-' tate ;(4:1)

F111 1' es cencc.

Benzene-cthyl ace- Do. (2:1)

Benzene-ethyl acetatc (1:1).

Slight -fluo rescencc.

I cu

, Benzene-ethyl acetote-(1:3).

,1 Benzene-ethyl aceate(154).

Benzene-ethyl race! tate 1:

Ethyl acetate Acetone ltlethanolinnnn Do; Do.

Certain of theresidues from the eluates from the ioregoing chromotogramwere crystallized from appropriate solvents (ethyl acetate, acetoneand-benzene, alone'or with 'neohexane) and the following'steroidswere"obtained? The column was' 'then eluted with benzene;benzene-ethyl acetate-mixtures; ethyl Fluores- M. P.,C.,rof' Emace W 25?crystalline wgghti Identification product 107 24 f186190,recryst; 101213. 5 Progesterone.

2 6.2 Do. 1.5 3.9 0.4 2.2 -17. 2 17-hydroxyprogesterone. 5.1 Do.

1.4 0.8 2. 2 IIB-hydroxyprogesterone. 4. 1 v Gorticosterone. 22-41 1 Do.

5:52 -D0.- 3" Mixture of corticosterone-and' lf-hydroxycorticosterone.115.5 196-201; 7..5 ,17-hydrox3tcort1costerone. 141 24.5 198-206 7.5 Do.197 .5-204 6 D0; 186-192 (from 6.5 Do. 142 16;5 motherliquol's from140-142 recryst.) 143 11.5 186-195 13.:5 ?17.-11yd1ioxycort1costerone"(impure) Examplelt 8 liters ofcitrated, filtered;:whole'ib.ovine bloodwere passed through tworatlivers.to;remouewfi brin. Then a solutionof'streptomycinand penicillin were added (as in Example12)*followedby 20m1. of a propylenev glycolrsolution containing 600-mgzofM-androstene-Za;lll dione. 'll'heresulting-solution was perfused throughfour Iacera-ted beef adrenal glands in .parallelusing venous entryasrin. Example. 12. The perfusion wasconducted over a periodof" 6.5hours, corresponding-- to 14 cycles of the perfusate medium. The.perfusa-te was then frozen toeffeet hemolysis, thawed,;and treated. withfourhportionsof activated charcoal (Dacro G-6O),SeachVWeighingAOt-g.Each treatment consisted in stirring the blood i'With, char- .coal forl-r-3 hours.-=at room temperature, allowing the charcoal tosettleovernight-at'roomtemperature, separatingsthe supernatant bloodby-decantation and centrifugation. Each batch of activated carbon wasthen-individually washed by centrifugationwith 1% saline solution,repeating this-washing;generally four onfiveir'timest Each of the fourcharcoal fractions above was stirrediwith acetone-,-ufiltered,landthefilter cake washed copiously with acetone. The filtrate from a each 10fthese: washings (called the 1 acetone extract) was concentrated: invacuo' to dryness. Thetresiduewas 1 then extracted-with boiling-ace toneand concentrated in vacuo 'to a -weighed residue; Each batch ofcharcoalwas washed copiously on the: filterwith -methylene:.chloride and! thenextractedr in 1 a 1 Soxhlet; apparatus* with methylene chloride for16424 hours; .then with vmethar-zol for-"3: 1 hours; Each: of. theextracts was: evaporated in vaeuo toia weighed-residue: The results areset'forth in the following table:

The residues from the first and second methylene chloride washes and thefirst and second methylene chloride extracts (weighing a total of 1400mg.) were combined, dissolved in benzene and passed over a columncontaining then extracted in a Soxhlet apparatus with -methylenechloride and finally with benzene, as in Example 12. Each of theextracts was evaporated and the residues were given the sulfuric acidtest with the following results:

V Charcoal 1," Charcoal 2 Charcoals 3-5nnd saline Extract 1 Hours Mg.H2504 Hours Mg. H2804 Hours Mg. H2804 Acetone (via chloroform) 103 194140 Trace Methylene chloride wash. 134 309 2. 75 339 Slight 50 g. ofsilica gel, as in Example 12. The column was then-developed and elutedas follows:

f Vol- Eluate Solvent ume, Product Inl.

Benzene-hexane 200 Syrup. Benzene 300 Do. Benzeneet-her (19: 200 M. P.52-53" C. Benzene-ether (9:1) 200 Syrup.

Benzene-ether (4:1) 400 Do.

Benzene-ether (3:1) 600 M. P. 131-132" C. Benzene-ether (2:1) 400 Do.Benzene-ether (1:1) 400 M. P. 225227 C. Benzene-ether (1:2 400 Do.Benzene-ether (1:3 200 M. P. 192-109 C.

Benzene-ether (1:4) 200 D0.

Ether 600 Do. Ether-ethyl acetate (19:1) 400 Do.

Example 14 To 8 liters of citrated, filtered, defibrinated, whole bovineblood containing penicillin and streptomycin as in Example 12 was addeda solution of 200 mg. of pregnenolone (A -pregnene- 3p-o1-20-one) in 15ml. of propylene glycol. The mixture was perfused through threelacerated .beef adrenal glands in parallel using venous entry for 200minutes, which corresponds to eight cycles of the fluid. The perfusatefluid (7700 m1., equivalent to 192 mg. of pregnenolone) was frozentoefiect hemolysis and then thawed. The blood was then treated with five40-g. portions of activated carbon (Darco G-GO) as in Example 12. Afterthe second charcoal treatment 5 g. of sodium fluoride were added tosuppress mold growth. Each of the five charcoal batches was washed withsaline solution and with acetone as in EX- ample 12. The saline washeswere combined and treated with activated charcoal (5 g. per liter). Theacetone washes were evaporated, the aqueou residues extracted withchloroform, and the chloroform extract of each batch was evaporated to aweighed residue. The batches of activated carbon were washed withmethylene chloride and The former was converted into androdo 34. 1Benzene-ethyl acetate (8:1). 18. 8 Benzene-ethyl acetate 1) 20. 2Benzene-ethyl acetate (4: 1) 67. 4 Ethyl acetate 516. 6

-do 12. 6 Acetone 19. 0

. do 36. 0 Methanol 61. 9

The total amount of solids obtained from these extracts was 5109.5 mg.This include 32 mg. of material giving a negative sulfuric acid test,which was obtained by extracting the filter papers and thimbles withbenzene.

The acetone extracts of the third, fourth, and fifth charcoals and thecharcoals from the saline washes and from all of the benzene extractswere combined, dissolved in benzene, and chromatographed over a columnof 80 g. of silica gel as in Example 12. The column was developed andeluted with benzene, benzene-ethyl acetate mixtures, acetone andmethanol with the following results:

Solvent Benzene Benenc-ethyl acetate (10:1) o...

Volume, Residue, Eluate I Solvent mg.

17 75 Benzene 8. 9 18 5O Benzene-ethyl acetate (10:1) 1. 5 19 50Bcnzeneethyl acetate (8:1) 6. 2 20-26.. 7X50 Benzene-ethyl acetate (6:1)313. 9 27-29.. 3X50 Benzene-ethyl acetate (4:1) 30. 0 30-35 3X50, 75,Benzene-ethyl acetate (3:1) 112. 9 36-39- 4X50 Benzene-ethyl acetate(2:1) 20. 8 4042 3X50 Benzene-ethyl acetate (1:1) 19. 2 4345 3X50Benzene-ethyl acetate (1:2) 10.8 40-421 3X50 Benzene-ethyl acetate (1:3)4. 7 49-51. 3X50 Ethyl acetate 5. 7 52. 5O Acetono 5. 3 53 100 Methanol12. 0

A third chromatogram was conducted on g. of silica gel using as thecharge all of the methylene chloride extracts of the first and secondbatches of activated carbon and residues 10 to 1? 1 ;;1 1Q 11 V of h fis h ma qe m in hi example. The following results were obtained:'

18 all. extrac (s of the remaining batches .of, charcoal.

The following results wereobtained:

Eluate Solvent Benzene Benzene-ethyl acetate (21:1) Benzene ethylacetate (10:1) Benzene-ethyl acetate (:1) Benzene-ethyl acetate (8:1)Benzene-ethyl acetate (7:1) Benzene-ethyl acetate (6:1) Benzene-ethylacetate (:1) Benzene-ethyl acetate (4:1) v Benzene-ethyl acetate (3:1)Benzene-ethyl acetate (2:1)

l3c nzene-ethyl acetate (1:1). Benzene-ethyl acetate (1:2).Benzene-ethyl acetate (1:3). Ethyl acetate Acetone... MethanolCrystalline materials were obtained from vari- Weight of Eluate gfSolvent residue 175 Benzene 135 2x100 Benzene-ethyl a tate 274.5 2x100Benzene-ethyl acetate (20:1) '11. 5 3x100 Benzene-ethyl acetate (:1) 1,.489.

100 .B enzene etliyl acetate (2:1) 137 100 Benzene-ethyl acetate (121)..189' 100 :Bcn'zcne-ethyl acetate (1:2).. 110 100 Benzene-ethyl acetate(1:3) 57. 4x100 Ethyl acetate. 189; 5

100 Acetone... 101 100 Methanol. ,133

A. second chromatogram was conducted psing 72 g, of silica. gel and theacetone and he methylene chlorideextracts of the first activated carbo Te fo ow n resul we obta nah ous eluates, using as crystallization mediaethyl acetate or acetone, alone or with neohexane. The Eluate 8 Solventrestate following results were obtained: (mg) 2 llgienzeneufilfl1.E..(..-..) 34.5 2 P. f enzene-c y aceta e 30:1 7 Residue We ght,Weight, Identity 2x50 Benzene-ethyl acetate (:1 4.5 v mg. tamed 0 Q 2-3X50 Benzene-ethyl acetate (10:1). 692-5 2X75 Benzene-ethyl acetate(9:1).. 51 2X75 Benzene-ethyl acetate (8:1).. 2 5 0. 5 2X75Benzene-ethyl acetate'(7: 1)... 11.5 16.8 167182 6.8 Oorticosterone.3x75 Benzene-ethyl a e .(6: 10.5 3 174.131 6, 2 D0 3X75 Benzene-ethylacetate (5:1; 13, 5 14' 2 175481 3. 2 15x75 Benzene-ethyl acetate (4:165619 17. 5 173-181 4 1) 11x75. Benzene-ethyl acetate (3:1) 68.8 19. 0195496 5. 2 Impure 17-hy- 5X75 Benzene-ethyl acetate (2:1) 237, 4droxycortico- 9X75 Benzene-ethyl acetate (1:1) --63."5 sterone. 9X75Benzene-ethyl acetatc (l:2) -70 5 117 13.8 191-196 3.7 Do. XBenzene-ethyl e e 1 2.3 118 11.7 106-197 3.2. l7-hydroxycortico- 2X75Benzene-ethy acetate (1P1) 5.0 t r 2X75 Benzene-ethyl acetate (1:6) 5, 2119 9.2 19%200 2.3 Do. g y a et te I th 1 .44. 152. 5-53 1.1 cc one. ,4N 3.521%. 7 1 3x100 Methano e2, 9 dues from 115-118. I 135. 15.1 320varlous crystalllneproducts were obtamed 136 0 g from the foregoingeluates using ethyl acetateas the crystallization solvent. The following-re- Example 15 sults were obtained:

8 liters of citrated, defibrinated, filtered, whole i' bovine bloodcontaining streptomycin and peni- 1M of cillin prepared as in Example 12was treated with Eluate gg g gg iggi li Identity asolution of 934 mg. ofl'l-hydroxyprogesterone 0 Q term in propylene glycol and then perfusedthrough e) four lacerated beef adrenaliglands in parallel for 1 7 7 a 1I 3 hours using venous entry. This corresponds i???- gg-g 7' lgio prpgsterone. to two actual cycles of the fluid. 7340 ml. of per- 50:: 17I220s -214v 112 D81 fusate fluid (corresponding to 852 mg. of 1'7- gghydroxyprogesterone) was frozen, thawed and 56316.1: c812, 193 --,1958:9 treated with 40-g. portions of activated charcoal 184 %QQ s3 8.4 186-1s9 3.2 at room temperature. The various charcoal preparations werewashed or extracted with ace- Z 16 tone, methylene chloride and benzeneas in Ex: mamp ample 12 with the following results. i '7 liters offiltered, defibrinated. whole boyine Charcoal 1 2 3 4 5 Saline Acetoneextract (via chloroform)-. 323 mg. 60.5 mg--.. 32 mg....- 88.5 mg.....100 mg..-- .34 mg. Fluorescence with cone. 112804.... vStrongPositive... Slight Negative.. Negative .Negative. Methylene chloridewash 392 mg mg-- 98n 1,...- 228 mg..';. 23lmg..'.; 18 mg.

Strong Positive... Positive... Pos 1tive Negative" Negative. 463mg...262 mg-- 337 mg..-. 404mg..- 517mg..-. 24 mg. Strong Negative.Negative;. Positive... Negative.- Negative. 44 mg...-.- 27.5 mg... 67.5mg, 59.5 mg."

Positive... Negative Slight.

Benzene extraction of all-.thimloles and filter pa- .pers yielded 18.5,mg. of. material which gave a slight fluorescence with concentratedsulfuric acid. The total extracted material, was 4064 mg.

. A chromatogram was conducted on 140, g. of silica gel as in Example 12using as the charge the benzene extract of thefirst bath of charcoal and.hydroxycorticosterone" blood were mixed with3 litersof citrated salinesolution (0.8% sodium chloride and.0.45% sodium citrate hydrate).'Then;1',O00,0 00.units otpenicillin G and 1 g. of streptomycin sulfatein saline solution are added.' To. this meo'iuniywas added a. solution1of 2 000 ,mg. of ll desoxyl'7- i 1 0 f 1 of. propy ene glycol. Thissolution was perfused through ten lacerated beef adrenal glands inparallel using venous entry for a total period of 7 hours, whichcorresponds to eleven cycles of the perfusion fluid. The citrated bloodwas removed from the perfusion apparatus and passed through a colloidmill to hemolyze the cells. 8.60 liters of the perfusion fluid(equivalent to 1720 mg. of ll-desoxy- 17-hydroxy-corticosterone) weretreated with three 50-g. portions of activated carbon (Darco G-60) as inExample 12. The three charcoal batches were combined, washed with salinesolution and then with a minimum of acetone to remove water. Thecharcoal was then extracted in a Soxhlet apparatus with 3 liters ofmethylene chloride for a period of 20 hours. The methylene chloridesolution was then evaporated in vacuo under nitrogen. The residue ofsteroidal material was crystallized from 50 cc. of ethyl acetate. Therewas obtained in this way 413 mg. of 17- hydroxycorticosterone whichmelts at 208 210 C.

The mother liquors from the foregoing crystallization were evaporated invacuo under nitrogen and the residual steroid mixture was taken up in150 ml. of benzene-ethyl acetate (9:1) and passed over a column of 80 g.of silica gel. The ,column was developed with 600 ml. of benzeneethylacetate (4:1) and eluted with 550 m1. of benzene-ethyl acetate (1:1),followed by 250 ml. benzene-ethyl acetate (2:3) and then 750 ml. ofethyl acetate. The eluates were evaporated junder nitrogen at reducedpressure, and the residues were crystallized from ethyl acetate. In thisway an additional crop of crystalline 17-hydroxycorticosterone, weighing327 mg., was obtained from the ethyl acetate eluate, as well as 240 mg.of ll-desoxy-l'l-hydroxycorticosterone from the benzene-ethyl acetate(1:1). The total yield of l'l-hydroxycorticosterone was 740 mg., whichis equivalent to 50% of theory based on the amount of starting materialconsumed.

Example 17 3 liters of citrated, filtered, defibrinated, whole bovineblood containing penicillin and streptomycin as in Example 12 weretreated with a solution of 600 mg. of ll-desoxycorticosterone in 45 ml.of propylene glycol. The resulting solution was perfused through eightlacerated beef adrenal glands for 4 hours (equivalent to 9.3 cycles).The blood was then hemolyzed by thawing and freezing. 2.88 liters of theperfusion liquid were treated with three 15-g. portions of activatedcarbon and the three batches of carbon were combined, washed with salineand a minimum of acetone and then extracted in a Soxhlet apparatus withmethylene chloride for hours. The methylene chloride extract wasevaporated in vacuo under nitrogen and the residue was taken up in '70cc. of benzene-ethyl acetate (9:1). This solution was passed over acolumn containing g. of silica gel. The column was developed with 250ml. of benzene-ethyl acetate (4:1). The column was then eluted with 250cc. of benzene-ethyl acetate (2:1) which removed most of thedesoxycorticosterone. This eluate was evaporated under vacuo innitrogave a greenish fluorescence were crystallized from ether andmaterial melting in the range of 160-170 C. was recovered ascorticosterone. Material melting in the range of -140 C. was recoveredas unchanged starting material. The column was finally eluted with 300cc. of ethyl acetate. This eluate was evaporated in vacuo under nitrogenand the residue was crystallized from a mixture of ethyl acetate andpetroleum ether. Three batches of crystalline corticosterone meltingrespectively at 169-1'72 0., 165168 Cs and -164 C. were obtained,weighing a total of 220 mg., which represents a yield of 41% of theoryon the basis of unrecovered starting material.

Example 18 300 mg. of 11-desoxy-17-hydroxycorticosterone 21-acetatedissolved in 10 ml. of propylene glycol was added to 3 liters ofcitrated whole bovine blood containing 300,000 units of penicillin G and300 mg. of streptomycin sulfate. The resulting fluid was perfusedthrough an intact beef adrenal gland, using arterial flow for 10 hours(one cycle). The blood was frozen, thawed, and treated successively withthree 15-g. portions of activated carbon. The activated carbon batcheswere combined, washed with saline solution and with a minimum ofacetone. The carbon was then extracted exhaustively in a Soxhletapparatus with methylene chloride for 20 hours. The methylene chlorideextract was evaporated in vacuo under nitrogen. The residue was taken upin benzene-ethyl acetate (9:1) and passed over a column of 18 g. ofsilica gel. The column was eluted with benzene-ethyl acetate and ethylacetate as in Example 16. From the ethyl acetate eluate was obtainedcrystalline 17-hydroxycorticosterone (25 mg.) melting at 208-210 C.after recrystallization from ethyl acetate. From the benzene-ethylacetate (1:1) eluate was obtained 110 mg. of 11-desoxy-1'7-hydroxycorticosterone.

We claim:

1. The method of hydroxylating a steroid which comprises addinga memberof the group of steroids having one of the following formulas:

CH: Z

and

CH; IY

gen and the residual steroid was recrystallized from ether, yielding 42mg. of desoxycorticosterone melting at 134-137" C. The column was theneluted with '15 cc. of benzene-ethyl acetate (1:1). This eluate wasevaporated in vacuo under nitrogen and the steroid residue was testedwith concentrated sulfuric acid. Residues which steroid may contain adouble bond at position 5, to a fluid selected from blood, bloodcomponents, blood substitutes and mixtures thereof with aqueous saltsolutions, perfusing a functioning, excised, mammalian adrenal glandwith said fluid, said adrenal gland being so incised as to penetratesuperficial layers without cutting the medullary layer and beingstructurally intact, and subsequently isolating the hydroxylatedsteroids from said fluid.

2. The method of claim 1 wherein the steroid is ll-desoxycorticosterone.

3. The method of claim 1 wherein the steroid is11-desoxy-17-hydroxycorticosterone.

4. The method of claim 1 wherein the steriod is progesterone.

5. The method of claim 1 wherein the steroid is A-androstene-3,17-dione.

6. The method of claim 1 wherein the steroid is l'l-hydroxyprogesterone.

7. The method of producing corticosterone which comprises perfusing afunctioning, excised, mammalian adrenal gland with blood plasmacontaining 11-desoxycorticosterone, said adrenal gland being so incisedas to penetrate superficial layers without cutting the medullary layerand being structurally intact, and subsequently isolating corticosteronefrom said plasma.

8. The method of producing 1'7-hydroxycorticosterone which comprisesperfusing a functioning,

excised, mammalian adrenal gland with blood plasma containing11-desoxy-l7-hydroxycorticosterone, said adrenal gland being so incisedas to penetrate superficial layers without cutting the medullary layerand being structurally intact, and subsequently isolating17-hydroxycorticosterone from said plasma.

9. The method of hydroxylating progesterone which comprises perfusing afunctioning, excised, mammalian adrenal gland with blood containingprogesterone, said adrenal gland being so incised as to penetratesuperficial layers without cutting the medullary layer and beingstructurally intact,

22 and subsequently isolating the hydroxylated steroids from said blood.

10. The method of producing 11-hydroxy-A andr0stene-3,l7-dione whichcomprises perfusing a functioning, excised, mammalian adrenal gland withblood containing A -androstene-3,17-dione, said adrenal gland being soincised as to penetrate superficial layers without cutting the medullarylayer and being structurally intact, and subsequently isolating11-hydroxy-A -androstene-3,l7 dione from said blood.

11. The method of producing 17-hydroxycorticosterone which comprisesperfusing a functioning, excised, mammalian adrenal gland with bloodcontaining 17-hydroxyprogesterone, said adrenal gland being so incisedas to penetrate superficial layers without cutting the medullary layerand being structurally intact, and subsequently isolating17-hydroxycorticosterone from said blood.

OSCAR HECI-ITER. ROBERT P. JACOBSEN. ROGER JEANLOZ. HAROLD LEVY. GREGORYPINCUS. VICTOR SCHENKER. CHARLES W. MARSHALL.

References Cited in the file of this patent UNITED STATES PATENTS NameDate Stern Nov. 18, 1924 OTHER REFERENCES Number

1. THE METHOD OF HYDROXYLATING A STEROID WHICH COMPRISES ADDING A MEMBEROF THE GROUP OF STEROIDS HAVING ONE OF THE FOLLOWING FORMULAS: